We studied the interaction of the androgen receptor with the MMTV promoter in living cells, and found a pronounced ligand effect on mobility of the receptor. We found that AR antagonists induce a high rate of exchange for the receptor with response elements, while agonist liganded receptors have a lower mobility. These results indicate that the longer residence time of the receptor is assoicated with a producitve promoter interation. Action of the glucocorticoid receptor (GR) was studied at the single cell level through the use of mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus. Direct binding of a GFP-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. A nonlinear relationship was observed between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells. We found that cochaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD), and examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Thus, activities impinging on the LBD regulate GR exchange with GREs, but the dissociation of GR from GREs is independent from ligand dissociation.